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New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of Oryctolagus Cuniculus

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New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of Oryctolagus Cuniculus International Journal of Molecular Sciences Article New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of Oryctolagus cuniculus Anna V. Glyakina 1,2, Alexey K. Surin 1,3,4, Sergei Yu. Grishin 1 , Olga M. Selivanova 1, Mariya Yu. Suvorina 1, Liya G. Bobyleva 5, Ivan M. Vikhlyantsev 5 and Oxana V. Galzitskaya 1,5,* 1 Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; [email protected] (A.V.G.); [email protected] (A.K.S.); [email protected] (S.Y.G.); [email protected] (O.M.S.); [email protected] (M.Y.S.) 2 Institute of Mathematical Problems of Biology, Russian Academy of Sciences, Keldysh Institute of Applied Mathematics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia 3 The Branch of the Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia 4 State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Moscow Region, Russia 5 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; [email protected] (L.G.B.); [email protected] (I.M.V.) * Correspondence: [email protected]; Tel.: +7-903-675-0156 Received: 29 September 2020; Accepted: 3 November 2020; Published: 6 November 2020 Abstract: To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed. Keywords: actin; monomer; filament; proteolysis; accessible surface area; mass spectrometry; electron microscopy 1. Introduction At present, much attention is being paid to the study of the mechanisms of aggregation of proteins and peptides, especially the elucidation of the organization of their ordered aggregates [1–4]. The study of the structure of protein homopolymers is associated with the problem of finding effective inhibitors of fibrillation in amyloid diseases [5–7]. Despite intensive research on this issue, there is currently no consistent model describing the molecular mechanism of amyloid fibril formation. Researchers face complex challenges; however, it is possible that the key factor preventing the construction of a unified model of fibril organization is the phenomenon of fibril polymorphism for the same protein or peptide [8–11]. Int. J. Mol. Sci. 2020, 21, 8319; doi:10.3390/ijms21218319 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 8319 2 of 15 Here we focus on actin, for which the filamentous form (F-actin) is its functional form [12–14]. F-actin is formed as a result of aggregation of the globular form of the actin monomer (G-actin), and this aggregation, in contrast to the formation of amyloids, is reversible [15–17]. The ability of actin to polymerize and depolymerize is of great importance for many biological functions, such as muscle contraction, cell migration, organization of the cytoskeleton, and transport of organelles [18–23]. The interaction of filamentous actin with myosin is the basis of muscle contraction. The protein titin plays a regulatory role in muscle contraction [24–27]. It was shown that an increase in Ca2+ concentration increases the strength and stability of the N2A region of interactions between titin and filamentous actin [28]. The length of actin filaments varies significantly depending on the type of tissue and localization in cells. For example, the length in sarcomeres is 1.10 0.03 µm, and the length in spectrin network ± in erythrocyte membrane is 33 5 nm [29,30]. There are about 160 different actin-binding proteins, ± the function of which is to block, stabilize, cross-link, and disrupt filaments [31,32]. Actin filaments are polar; their ends (slow-growing and fast-growing) differ in the structure and dynamics of polymerization/depolymerization. For the fast-growing end, the capping proteins are gelsolin, CapZ, and adducin [31,33,34], and for the slow-growing end, tropomodulin [35], acumentin [36], and Arp2/3 complex [37]. Tropomodulin has been found in various tissues and cells, and its role is especially important when actin filaments must maintain a constant length [38]. Since the 1950s, intensive studies have been carried out on the structure of monomeric and filamentous actin. Based on the data of X-ray structural analysis of polymer actin, it was suggested that F-actin can be helical [39]. On the basis of EM analysis and data from paper [39], it was first stated that F-actin is a double helix [40]. Since then, the idea of a double-stranded organization of filamentous actin has been generally accepted. The atomic structure of F-actin in the form of a double helix was proposed by Holmes et al. in 1990 [41] based on the fitting of the crystal structure of monomeric G-actin in the X-ray diffraction data obtained for oriented F-actin gels [41]. In this case, it was assumed that the structures of F-actin and actin in combination with DNase I are the same. Two actin molecules interact with each other at the following amino acid residues: 322–325 with 243–245, 286–289 with 202–204, 166–169 and 375 with 41–45. It has also been shown that modification of the amino acid residue H40 prevents the formation of filamentous actin. In the subsequent years, this model was refined and verified [18,42,43]. In the work of Kudryashov et al. [42] the structure of filamentous actin was proposed on the basis of dimeric crystal structures, which were obtained by cross-linking of amino acid residues Q41 and C374 included in two adjacent actin molecules. This model differs from the Holmes ‘s model [41] by the smaller twist of two consecutive actin molecules relative to each other. Consequently, there is a decrease in the distance (from 20 to 3 Å on average) between the following amino acid residues: E205 (OE2) and K291 (NZ), S199 (O) and K291 (NZ), T203 (OG1) and D288 (OD2), G197 (O) and T324 (CG2), S199 (CB) and T324 (OG1), T202 (CG2) and I287 (O), D244 (CB) and K326 (NZ). The structure proposed by Oda et al. [18] suggests that the conformational transition of actin from a monomeric to a filamentous form occurs by a simple rotation of two adjacent actin molecules relative to each other by 20◦, which makes actin fibril flat. Oztug Durer et al. [43] showed that the cross-linking of amino acid residues 45 and 169, 47 and 169, and 50 and 169 leads to the destruction of F-actin. The structures of filamentous actin in the double-stranded form listed above may not be entirely correct. It was also shown that archaeal filamentous actin has a single-stranded helical structure [44,45], but bacterial filamentous actin [46], like eukaryotic, has a double-stranded helical structure. It was found that archaeal actin (crenactin) is more similar in amino acid sequence to eukaryotic than to bacterial actin. Usually, in all these models of filamentous actin (eukaryotic, bacterial and archaeal) monomers do not overlap with each other. The strong polymorphism of F-actin [47–51] justifies the continuation of studies of the morphology of filaments in coordination with alternative interpretations of the structural features of F-actin. It was shown in [51] that actin structures can be divided into four large groups (F-, C-, O-, and G-forms) based on the orientation of two main domains: the outer domain and the inner domain. The outer domain Int. J. Mol. Sci. 2020, 21, 8319 3 of 15 consists of subdomains 1 and 2, and the inner domain consist of subdomains 3 and 4. The F-form was observed in the structures of naked actin and actin filaments in complex with tropomyosin. The C-form was observed in actin filaments decorated with cofilin. The O-form was observed in phosphate-treated crystals of the profilin-actin complexes. The G-form has been observed in the crystal structures of monomeric actin. The F-form and C-form cannot transform to the G-form by thermal fluctuations. So, extensive literature data show that F-actin cannot be described by a single structural model, since this model cannot explain the introduction of cross-links (S-S bonds) inside the filament. These results indicate a high degree of plasticity and heterogeneity of F-actin. Int.Int. J. J. Int.Mol. Mol. J. Int.Sci. Sci.Mol. J.2020 2020 Mol.Sci.Int., ,202021 21Sci. J., ,Mol.x x , 2020 FOR21FOR ,Sci. x, PEER21FORPEER 2020, x FORPEER REVIEW ,REVIEW 21, PEERx REVIEW FOR REVIEW PEER REVIEW 33 ofof 16316 of 316 of 163 of 16 Our latest preliminary experimental data for limited proteolysis, mass spectrometry analysis, X-raybyby di thermal thermalffbyraction, thermalby fluctuations. thermalfluctuations.by andfluctuations. thermal fluctuations. electron So, So, fluctuations. extensive extensive So, microscopy extensive So, extensive literature literature So, extensiveliterature literature images data data dataliteratureshow show aredata show that that inconsistent show data F thatF‐‐actinactin thatshow F‐actin cannot cannot F ‐thatactin cannot with Fbe be ‐cannotactin described described be the cannotdescribed be double described by by be a a describedbysingle single helical a by single a single organizationby a single structural model,structural since this model, model since cannot this modelexplain cannot the introduction explain the of introduction cross‐links (Sof‐ Scross bonds)‐links inside (S‐S bonds) inside of filamentousstructuralstructuralstructural model, actin.
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